Compounds and related methods for inhibition of gamma-aminobutyric acid aminotransferase

ABSTRACT

(1S, 3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic acid illustrates a novel class of compounds as potent irreversible inhibitors of γ-aminobutyric acid aminotransferase (GABA-AT). The corresponding monofluoro-substituted compounds also are potent time-dependent inhibitors of GABA-AT.

This application is a continuation of and claims priority benefit fromapplication Ser. No. 10/623,152 filed Jul. 18, 2003, issued as U.S. Pat.No. 6,794,413 on Sep. 21, 2004, and provisional patent application Ser.No. 60/397,498 filed Jul. 19, 2002, each of which is incorporated hereinby reference in its entirety.

The United States government has certain rights to this inventionpursuant to Grant No. NS 15703 from the National Institutes of Health toNorthwestern University.

BACKGROUND OF THE INVENTION

Epilepsy is an important central nervous system disease characterized byrecurring convulsive seizures. It has been shown that convulsions arisewhen an imbalance exists between two principal neurotransmitters,L-glutamic acid, an excitatory neurotransmitter, and γ-aminobutyric acid(GABA), an inhibitory neurotransmitter. Two enzymes, glutamic aciddecarboxylase (GAD) and γ-aminobutyric acid aminotransferase (GABA-AT),regulate the level of GABA in the brain. GAD synthesizes GABA fromL-glutamic acid. GABA-AT, which is a pyridoxal-5′-phosphate (PLP, 1)dependent enzyme, converts GABA to succinic semialdehyde. Duringtransamination, PLP is converted to pyridoxamine-5′-phosphate (PMP),which is transformed back to PLP by a reaction with 2-ketoglutarate; the2-ketoglutarate is converted to L-glutamic acid. Succinic semialdehydedehydrogenase (SSDH) oxidizes succinic semialdehyde to succinic acidusing NADP⁺ as the cofactor (Scheme 1).

When the level of GABA in the brain falls below a threshold level,convulsions occur. Taking GABA orally is not effective in raising theGABA level in the brain because GABA cannot cross the blood-brainbarrier. One promising approach is to inhibit GABA-AT, which degradesGABA. The level of GABA would increase due to its continuous production.The catalytic mechanism of GABA-AT is shown in Scheme 2. The cofactorPLP is bound to Lys329 in the form of a Schiff base. (Storici, P.;Capitani, G.; Biase, D. D.; Moser, M.; John, R. A.; Jansonium, J. N.;Schirmer, T. Crystal Structure of GABA-Aminotransferase, a Target forAntiepileptic Drug Therapy. Biochemistry, 1999, 38, 8628-8634.)Transimination gives the imine between GABA and PLP. The enzyme thenremoves the γ-proton of GABA to give the aldimine, which is subsequentlyhydrolyzed to produce succinic semialdehyde and PMP.

In the art, it is understood by definition that a mechanism-basedirreversible inhibitor is an unreactive compound that has a structuralsimilarity to the substrate or product for the target enzyme and isconverted by the target enzyme into a species that inactivates theenzyme prior to its release from the active site. (Silverman, R. B.Mechanism-Based Enzyme Inactivation: Chemistry and Enzymology, Vol. 1;CRC Press: Boca Raton, 1988.) One such inhibitor is the rationallydesigned vigabatrin (2), an epilepsy drug marketed all over the world,except in the U.S., which irreversibly inhibits GABA-AT by themechanisms shown in Scheme 3. Pathway a (Michael addition) has beendetermined and found to account for about 70-75% of the totalinactivation. (Nanavati, S. M.; Silverman, R. B. Mechanisms ofInactivation of γ-aminobutyric Acid Aminotransferase by the AntiepilepsyDrug γ-Vinyl GABA (Vigabatrin). J. Am. Chem. Soc. 1991, 113, 9341-9349.)

Previously, 3, a conformationally-rigid vigabatrin analogue, wassynthesized.

Surprisingly, 3 was not a GABA-AT inactivator but was a very goodsubstrate (K_(m)=0.1 mM, k_(cat)=11.7 min⁻¹, k_(cat)/K_(m)=117 mM⁻¹min⁻¹) with a specificity constant almost six times greater than that ofGABA (K_(m)=2.4 mM, k_(cat)=49 min⁻¹, k_(cat)/K_(m)=20.4 mM⁻¹ min⁻¹). Itwas later determined by computer modeling that the endocyclic doublebond is not in the right orientation for Michael addition (pathway a,Scheme 3), nor is it an effective enamine for enzyme inactivation.Therefore 7, which has an exocyclic double bond, was designed andprepared from diketone 4, as shown in Scheme 4. (Qiu, J.; Pingsterhaus,J. M.; Silverman, R. B. Inhibition and Substrate Activity ofConformationally Rigid Vigabatrin Analogues with γ-Aminobutyric AcidAminotransferase. J. Med. Chem. 1999, 42, 4725-4728.) An additionreaction with (trimethylsilyl)methylmagnesium chloride followed byelimination furnished 6. Deprotection of the benzyl group and hydrolysisof the lactam gave the amino acid 7. (Specific reagents and conditions:(a) TMSCH₂MgCl, −30° C. to RT, 38%; (b) (CF₃CO)₂O, DMAP, then TBABr, KF,86%; (c) Na/NH₃/^(t)BuOH,; (d) 2N HCI, 90%, 2 steps.)

Interestingly, 7 inactivated GABA-AT, but when 2-mercaptoethanol wasadded to the incubation mixture, no inactivation was observed. Apossible mechanism accounting for this phenomenon is shown in Scheme 5.It is likely that 7 is only a substrate for GABA-AT. After formation of8, the double bond is not reactive enough, so this intermediate is nottrapped by the enzyme, but rather is released from the active site inthe form of an α,β-unsaturated ketone (9). In the presence of2-mercaptoethanol, a reactive nucleophile, 9 is trapped to form 10,giving no inactivation

of the enzyme. In the absence of 2-mercaptoethanol, however, 9 mayreturn to the enzyme and become covalently attached to the enzyme (11),leading to the enzyme's inactivation. According, however, todefinitions, prior art compound 7 is not a mechanism-based inactivatorinasmuch as inactivation does not occur prior to the release of theactive species from the active site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically compares activities of vigabatrin (♦) and adifluoro-substituted compound (▪), 15, in accordance with thisinvention. For vigabatrin: y=6.0295x+1.8585, and R²=0.9553. For compound15: y=0.1227x+3.948, and R²=0.9965.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide various compounds and/or compositions and related methodologiesfor the inactivation and/or inhibition of γ-aminobutyric acidaminotransferase, such inactivation or inhibitory activity as can beused in the treatment of epilepsy and other CNS disease states,including addiction, thereby overcoming various deficiencies andshortcomings of the prior art, including those outlined above. It willbe understood by those skilled in the art that one or more aspects ofthis invention can meet certain objectives, while one or more otheraspects can meet certain other objectives. Each objective may not applyequally, in all its respects, to every aspect of this invention. Assuch, the following objects can be viewed in the alternative withrespect to any one aspect of this invention.

It is an object of the present invention to provide a mechanism-basedinhibition and/or inactivation methodology and one or more compoundsuseful in conjunction therewith.

It is another object of the present invention to provide one or morecompounds demonstrating inhibitory activity with respect toγ-aminobutyric acid aminotransferase, such activity as would beunderstood by those skilled in the art to be efficacious in thetreatment of epilepsy and addiction.

It is another object of the present invention to provide one or morecompounds incorporating rationally-designed structural characteristicsconsistent with mechanism-based inhibition/inactivation ofγ-aminobutyric acid aminotransferase, such compounds having incorporatedtherein an electron-deficient methylene moiety for irreversibleinteractive contact with an active site of such an enzyme.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofcertain embodiments, and will be readily apparent to those skilled inthe art having knowledge of various enzyme systems, and inhibitorycompounds and their preparation. Such objects, features, benefits andadvantages will be apparent from the above as taken into conjunctionwith the accompanying examples, data, FIGURES and all reasonableinferences to be drawn therefrom.

In part, the present invention comprises a γ-aminobutyric acidaminotransferase inhibitor compound of a formula

wherein R₁ and R₂ are selected from H and F, and at least one of R₁ andR₂ is F. Alternatively, such inhibitors can be salts of such a compound;that is, including but not limited to an ammonium salt or a carboxylatesalt of such a compound. Whether monofluoro- or difluoro-substituted,the amino and carboxy substituents can have either a cis or transstereochemical relationship. In monofluoro-substituted embodiments, suchcompounds can have either a Z or an E configuration.

As shown below in the context of several synthetic preparations, certainembodiments of the present inhibitor compounds can be provided as anammonium salt, wherein the counter ion is the conjugate base of a proticacid. Likewise, in various other embodiments, such inhibitor compoundscan be provided and/or utilized as a carboxylate, in conjunction, forexample, with a corresponding ammonium (e.g., +NHR₃, where R is hydrogenor alkyl), alkaline or alkaline-earth cation. Without limitation,certain preferred embodiments of this invention can comprise monofluoro-or difluoro-substituted compounds as either the ammonium hydrochloridesalt or sodium carboxylate.

As discussed more fully below in conjunction with known and acceptedmechanistic considerations, the present invention can also include acomplex comprising the addition product of a γ-aminobutyric acidaminotransferase and a compound of this invention, such a complexinactivating the enzyme component thereof. Without limitation, suchcompounds include those discussed more fully above and illustratedbelow, all as can be varied in accordance within the range ofstereochemical and/or configurational relationships contemplated withinthe broader aspects of this invention. As would be understood by thoseskilled in the art, the enzyme component of such an addition productfurther comprises a pyridoxal-5^(/)-phosphate cofactor.

Accordingly, the present invention also includes a method of inhibitinga γ-aminobutyric acid aminotransferase. Such a method comprisescontacting the enzyme with at least a partially effective amount of oneof the aforementioned inventive compounds. Such contact would be asunderstood by those skilled in the art experimentally and/or forresearch purposes or as may be designed to simulate one or more in vivoor physiological conditions. In certain embodiments, an addition can beachieved with one or more difluoro-substituted compounds. In otherembodiments, monofluoro-substituted compounds can be used withcomparable effect. Regardless of fluoro-substitution, the amino andcarboxy substituents can vary by degree of protonation and the presenceas the corresponding salt. Likewise, such compounds are considered overthe full range of stereochemical and/or configurational isomers.

Moreover, in yet another departure from the prior art, the presentinvention provides a method of using an electron-deficient exocyclicmethylene moiety to inhibit γ-aminobutyric acid aminotransferaseactivity. Such a method comprises (1) providing a compound from a groupof compounds of a formula

wherein R₁ and R₂ are selected from H and F, and at least one of R₁ andR₂ is F; such compounds including salts thereof; and (2) contacting sucha compound with a γ-aminobutyric acid aminotransferase, the exocyclicmethylene moiety of the compound capable of binding to an active siteresidue of the enzyme. Without limitation, such compounds are eithermonofluoro- or difluoro-substituted, and can vary within the full rangeof structural, ionic, stereochemical and/or configurationalconsiderations discussed above. Nonetheless, certain cis and transisomers are used, as provided in the following examples, to demonstrateone or more aspects regarding the utility of this invention.

Because prior art methylene compound 7 did not inactivate GABA-AT, anapproach was taken to design the compounds of this invention, inparticular the more reactive, difluoro-substituted compound 15. Withoutlimitation to any one theory or mode of operation, the comparable sizebut higher electronegativity of fluorine, as compared to hydrogen,provides a much more reactive intermediate. Prior to its release, thisspecies may be sufficiently reactive to become covalently attached orbound to GABA-AT upon contact or reaction therewith, leading to enzymeinactivation. Illustrating such embodiments, compound 15 was preparedfrom 12 (Scheme 6). Compound 13 was prepared by aHorner-Wadsworth-Emmons reaction. (Piettre, S. R.; Cabanas, L.Reinvestigation of the Wadsworth-Emmons Reaction Involving LithiumDifluoromethylenephosphonate. Tetrahedron Lett. 1996, 37, 5881-5884.) Itwas then deprotected using ceric ammonium nitrate (CAN) to give 14 andhydrolyzed to give 15. (Qiu, J.; Silverman, R. B. A New Class ofConformationally Rigid Analogues of 4-Amino-5-halopentanoic Acids,Potent Inactivators of γ-Aminobutyric Acid Aminotransferase. J. Med.Chem. 2000, 43, 706-720.) (See, more particularly, examples 5-7, below.)

Compound 15 was found to be a very potent GABA-AT inactivator, even inthe presence of 2 mM 2-mercaptoethanol. Because of its high potency,k_(inact) and K_(I) values could not be determined accurately underoptimal conditions (pH 8.5, 25° C.), where the enzyme exhibits maximumactivity. Comparisons were made with (S)-vigabatrin of the prior art.Taking only the first two data points for both 15 and (S)-vigabatrin,the k_(inact)/K_(I) value for 15 is 186 times greater than that for(S)-vigabatrin. In order to obtain more accurate data, 15 and vigabatrinwere compared under nonoptimal conditions. Even at 0° C. in pH 8.5buffer, no pseudo first-order kinetics were observed for 15. At pH 6.5(25° C.) 15 (K_(I) 31 μM, k_(inact) 0.18 min⁻¹, k_(inact)/K_(I) 5.7mM⁻¹min⁻¹) is 50 times more potent than (S)-vigabatrin (K_(I) 3.24 mM,k_(inact) 0.37 min⁻¹, k_(inact)/K_(I) 0.114 mM⁻¹min⁻¹). (See alsoexample 14 and FIG. 1.) The exocyclic methylene moiety and/or the rigidconformation of 15, which is believed to minimize the entropic penaltyon binding, may contribute to its potency. Fluorine incorporation mayalso make a corresponding intermediate sufficiently reactive to betrapped by the enzyme. While the cis isomer is shown in Scheme 6.Comparable results can be obtained with the trans isomer, as can beprepared through a straight-forward extension of the synthetictechniques described herein, as would be understood by those skilled inthe art.

Likewise, this invention contemplates various monofluoro-substitutedcompounds. The syntheses of compounds 20 and 22 are shown in Scheme 7.The reaction of prior art starting material 12 withfluoromethylphenylsulfone and diethylphosphoryl chloride gave 16 as amixture of the two isomers, which was then subjected to the reductionwith magnesium and mercury chloride, giving 17 and 18 which wereseparated and isolated. Further deprotection of the lactam thenhydrolysis gave 20 and 22. (See examples 8 and 13, below.) Consistentherewith, compounds 20 and 22 also are potent time-dependent inhibitorsof GABA-AT. Similar activities can be demonstrated with thecorresponding trans isomers.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compounds and/methods of the presentinvention, including the preparation of molecular compounds havingvarious structural moieties for inhibitory interaction with aγ-aminobutyric acid aminotransferase, such compounds as are availablethrough the synthetic methodologies described herein. In comparison withthe prior art, the present methods and compounds provide results anddata which are surprising, unexpected and contrary to the prior art.While the utility of this invention can be illustrated through the useof several compounds and structural moieties incorporated therein, itwill be understood by those skilled in the art that comparable resultsare obtainable with various other compounds and moieties consistentherewith and as are commenced right with the sculpt of this invention.

General Methods. All NMR spectra were recorded on either a VarianMercury 400 MHz or a Varian Inova 500 MHz NMR spectrometer. ¹H chemicalshifts are reported as δ values in ppm downfield from Me₄Si as theinternal standard in CDCl₃. For samples run in D₂O, the HOD resonancewas set at 4.80 ppm. ¹³C chemical shifts are listed in ppm with theCDCl₃ carbon peak set to 77.23 ppm. For samples run in D₂O, DSS was usedas the external standard. ¹⁹F chemical shifts are listed in ppm withCFCl₃ as the external standard for samples run in CDCl₃ and TFA as theexternal standard for samples run in D₂O. Mass spectra were obtained ona VG70-250SE mass spectrometer. Column chromatography was carried outwith Merck silica gel 60 (230-400 mesh ASTM). TLC was run with EMScience silica gel 60 F254 preloaded glass plates. Cation-exchange resinwas purchased from Bio-Rad Laboratories. An Orion Research 702 pH meterwith a general combination electrode was used for pH measurements. Allenzyme assays were recorded on a Perkin-Elmer Lambda 10 UV/Visspectrometer.

Reagents. Fluoromethyl phenylsulfone was purchased from TCI America,Inc. All other reagents were purchased from Aldrich Chemical Co. andwere used without purification. All solvents were purchased from FisherScientific. Anhydrous THF was distilled from sodium metal undernitrogen.

Example 1

6-Oxo-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (4).6-exo-Acetoxy-2-benzyl-7-anti-bromo-2-azabicyclo[2.2.1]heptan-3-one(2.15 g, 6.6 mmol) was added to a solution of tributyltin hydride (2.9g, 9.9 mmol) and AIBN (20 mg) in anhydrous benzene (20 mL). (Qiu, J.;Silverman, R. B. A New Class of Conformationally Rigid Analogues of4-Amino-5-halopentanoic Acids, Potent Inactivators of γ-AminobutyricAcid Aminotransferase. J. Med. Chem. 2000, 43, 706-720.) The resultantsolution was heated at reflux and stirred for 12 h. After the solutionwas concentrated under reduced pressure, the residue was purified byflash column chromatography on silica gel, eluting with ethylacetate/hexane (2:3), to afford6-exo-acetoxy-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (1.37 g, 85%) asa colorless oil. ¹HNMR (300 MHz, CDCl₃) 7.22 (5H, m, ArH), 4.76 (1H, m,H₆), 4.65 (1H, d, J, 15 Hz, ArCH₂), 4.01 (1H, d, 15 Hz, ArCH₂), 3.67(1H, m, H₁), 2.80 (1H, m, H₄), 1.80-2.20 (4H, m, H₅ and H₇), 2.01 (3H,s, CH₃CO₂); m/z (EI) 260, 216, 173, 91, 65; HRMS (EI) calcd forC₁₅H₁₇NO₃ M 259.1208, found M 259.1210.

6-exo-Acetoxy-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (1.2 g, 4.6 mmol)was added to a stirred suspended solution of K₂CO₃ (1.9 g, 13.8 mmol) inmethanol (15 mL) and water (5 mL). After being stirred for 3 h, thereaction mixture was concentrated under reduced pressure. The resultantaqueous layer was extracted with ethyl acetate (3×30 mL). The organiclayer was washed with brine (25 mL) and dried over anhydrous Na₂SO₄. Thesolvent was concentrated under reduced pressure to afford6-exo-hydroxy-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (0.99 g, 99%) asa solid. ¹HNMR (300 MHz, CDCl₃) 7.20 (5H, m, ArH), 4.60 (1H, d, J, 15Hz, ArCH₂), 4.04 (1H, d, 15 Hz, ArCH₂), 4.00 (1H, m, H₆), 3.54 (1H, m,H₁), 2.77 (1H, m, H₄), 1.5-2.2 (4H, m, H₅ and H₇); m/z (EI) 217, 173,144, 91, 65; HRMS (EI) calcd for C₁₃H₁₅NO₂ M 217.1103, found M 217.1104.

4-Methylmorpholine N-oxide (0.77 g, 6.6 mmol), tetrapropylammoniumperruthenate (TPAP) (5 mg) and 4 Å sieves were added to a stirredsolution of 6-exo-hydroxy-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (0.67g, 3.3 mmol) in anhydrous CH₂Cl₂ (10 mL). After being stirred for 14 h,the mixture was concentrated under reduced pressure. The resultantslurry was loaded on the flash silica gel column directly, eluting withEtOAc/hexane (2:3), to afford 4 (0.5 g, 75%) as a colorless solid. ¹HNMR(300 MHz, CDCl₃) 7.30 (5H, m, ArH), 4.80 (1H, d, J, 15.1 Hz, ArCH₂),3.95 (1H, d, 15 Hz, ArCH₂), 3.60 (1H, m, H₁), 3.07 (1H, m, H₄),2.33-2.20 (3H, m, H₅ and H₇), 1.90 (1H, d, 10 Hz, H₅); m/z (EI) 215,187, 132, 91, 65; HRMS (EI) calcd for C₁₃H₁₃NO₂ M 215.0946, found M215.0949.

Example 2

6-exo-Trimethylsilylmethyl-6-endo-hydroxyl-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one(5). (Trimethylsilylmethyl) magnesium chloride (1M, 3.7 mL, 3.7 mmol)was injected to a stirred solution of6-oxo-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (4) (0.50 g, 2.5 mmol) inanhydrous THF (10 mL) at −30° C. under N₂ protection. After beingstirred for 5 h at −30° C., the reaction mixture was stirred overnightat room temperature. The reaction mixture was treated with saturatedNH₄Cl (20 mL). The resultant aqueous layer was extracted with ethylacetate (3×25 mL). The combined organic layers were washed with water(20 mL), brine (25 mL), dried over anhydrous Na₂SO₄, and concentratedunder reduced pressure. The residue was purified by flash columnchromatography on silica gel, eluting with ethyl acetate/hexane (1:3),to afford 5 (0.23 g, 38%) as a colorless solid. ¹HNMR (300 MHz, CDCl₃)7.33 (5H, m, ArH), 5.14 (1H, d, J, 15.1 Hz, ArCH₂), 4.07 (1H, d, J 15Hz, ArCH₂), 3.33 (1H, t, J 1.6 Hz, H₁), 2.77 (1H, dd, J 3.6, 1.6 Hz,H₄), 2.03-1.67 (4H, H₅ and H₇), 1.12 (1H, d, J 3.3 Hz, CH₂Si), 0.11 (9H,m, Si(CH₃)₃); ¹³CNMR (300 MHz, CDCl₃) 177.1, 137.8, 128.9, 128.3, 127.6,83.1, 68.7, 47.3, 46.0, 43.4, 41.0, 31.1, 0.64; m/z (EI) 303, 189, 173,145, 91, 73; HRMS (EI) calcd for C₁₇H₂₅NO₂Si M 303.1655, found M303.1657.

Example 3

6-Methylenyl-2-benzyl-2-azabicyclo[2.2.1]heptan-3-one (6).4-Dimethylaminopyridine (1.71 g, 14 mmol) and trifluoroacetic anhydride(1.47 g, 7.0 mmol) were added to a stirred solution 5 (0.21 g, 0.7 mmol)in anhydrous THF (10 mL) at room temperature. After the reaction mixturewas stirred for 3 h, tetrabutylammonium bromide (3.38 g, 10.5 mmol) andpotassium fluoride (1.22 g, 21 mmol) were added to the reaction mixture.The resultant mixture was stirred for 15 h at 50° C. The reactionmixture was diluted with saturated NH₄Cl (25 mL) and the aqueous layerwas extracted with ethyl acetate (3×40 mL). The combined organic layerswere washed with water (25 mL) and brine (25 mL), and dried overanhydrous Na₂SO₄, and concentrated under reduced pressure. The residuewas purified by flash column chromatography on silica gel, eluting withethyl acetate/hexane (1:3), to afford 6 (0.13 g, 86%) as a colorlesssolid. ¹HNMR (300 MHz, CDCl₃) 7.33 (5H, m, ArH), 5.05 (1H, s, C═CH₂),4.95 (1H, s, C═CH₂), 4.79 (1H, d, J, 15.3 Hz, ArCH₂), 3.75 (1H, s, H₁),3.72 (1H, d, J 15.3 Hz, ArCH₂), 2.92 (1H, dd, J 1.2, 2.7 Hz, H₄), 2.44(1H, m, H_(5exo) or H_(5endo)), 2.24 (1H, m, H_(5exo) or H_(5endo)),2.00 (1H, m, H_(7anti) or H_(7syn)) 1.56 (1H, m, H_(7anti) or H_(7syn)),¹³C NMR (300 MHz, CDCl₃) 196.1, 178.6, 146.2, 137.4, 128.8, 128.3,127.6, 107.4, 64.4, 54.7, 44.3, 40.4, 31.9; m/z (EI) 213, 172, 106, 91;HRMS (EI) calcd for C₁₅H₁₅NO M 213.1154, found M 213.1162.

Example 4

3β-Amino-4-methylenylcyclopentane-1β-carboxylic acid (7). Freshly-cutpieces of sodium (0.16 g) were added to a stirred solution of liquid NH₃(8 mL) and t-BuOH (2 mL) at −78° C. to afford a deep blue solution.Then, a solution 6 (0.11 g, 0.51 mmol) in THF (8 mL) was stepwise addedto the stirred sodium-liquid ammonium solution at −78° C. After theresulting solution was stirred for 10 min at −78° C., the solution wasraised to −30° C. and stirred for 4 min. Then, the solution was cooledback to −78° C. Acetic acid (2 mL) was added slowly. After the solutionwas allowed to warm to room temperature, the resultant slurry wasfiltered and washed with ethyl acetate (100 mL). The organic solutionwas concentrated under reduced pressure to afford a solid residue.Without any purification, the residue was added to a stirred solution ofacetic acid (5 mL) and 4N HCl (5 mL). The resulting solution was stirredat 70° C. for 5 h, and concentrated in vacuo to give a solid. The solidwas purified by ion-exchange chromatography (AG® 50W-X8), eluting withwater and 0.5 M NH₄OH, to afford 7 (0.065 g, 90%) as a colorless solid.¹HNMR (300 MHz, D₂O) 5.15 (1H, m, C═CH₂), 5.07 (1H, m, C═CH₂), 3.97 (1H,m, H₃), 2.61 (1H, m, H₁), 2.68-2.39 (2H, m, H₂), 2.20 (1H, ddd, J 14.2,7.2, 7.0 Hz, H_(5a) or H_(5β)), 1.73 20 (1H, ddd, J 14.2, 7.2, 7.0 Hz,H_(5a) or H_(5β)); m/z (EI) 141, 96, 69; HRMS (EI) calcd for C₇H₁₁O₂N M141.0790, found M 141.0787, CHN analysis calcd for C₇H₁₁O₂N, H % 7.85 C% 59.56 N % 9.92, found H % 7.88 C % 59.23 N % 9.62.

Example 5

(1S,4S)-6-Difluoromethylenyl-2-(4′-methoxybenzyl)-2-azabicyclo[2.2.1]heptan-3-one(13). At −78° C., ^(t)BuLi (1.7 M in pentane, 1.73 mL, 2.94 mmol) wasslowly added to a stirred solution of diethyl(difluoromethyl)phosphonate (0.48 mL, 2.94 mmol) in anhydrous THF (15mL). After being stirred for 0.5 h at −78° C., 12 (0.60 g, 2.45 mmol) inanhydrous THF (20 mL) was slowly added via syringe. Stirring continuedfor 1 h at −78° C., then the solution was allowed to warm to roomtemperature and heated to reflux for 24 h. Compound 12 is known andavailable in the art, and can be prepared as described in Qiu, J.;Silverman, R. B. A New Class of Conformationally Rigid Analogues of4-Amino-5-halopentanoic Acids, Potent Inactivators of γ-AminobutyricAcid Aminotransferase. J. Med. Chem. 2000, 43, 706-720. After thereaction had cooled down, THF was evaporated, and saturated NH₄Clsolution (20 mL) was added to the residue, which was extracted withEtOAc (3×20 mL). The organic layer was washed with brine (2×20 mL),dried over anhydrous Na₂SO₄, and concentrated under reduced pressure.The residue was purified by flash column chromatography, eluting withhexanes/ethyl acetate (2:1) to give 13 (0.47 g, 68%) as a colorless oil:¹H NMR (400 MHz, CDCl₃) δ 7.18 (d, J 8.4 Hz, 2H), 6.07 (d, J 8.4 Hz,2H), 4.63 (d, J 14.8 Hz, 1H), 4.14 (s, 1H), 3.80 (s, 3H), 3.78 (d, J14.8 Hz, 1H), 3.00 (s, 1H), 2.50 (dt, J 15.2, 3.6 Hz, 1H), 2.27 (dd, J15.2, 2.4 Hz, 1H), 2.00 (d, J 9.2 Hz, 1H), 1.53 (d, 9.6 Hz, 1H); ¹³C NMR(100 MHz, CDCl₃) δ 177.37, 159.13, 152.19 (dd, J 285.7, 281.2 Hz),129.59, 128.47, 114.13, 88.95 (dd, J 25.6, 22.2 Hz), 58.38 (d, J 5.3Hz), 55.50, 45.60, 44.59, 40.96, 27.43; ¹⁹F NMR (376 MHz, CDCl₃) δ 42.64and 41.01 (2 dd, J 60.2, 2.3 Hz, 2F). HRMS (EI) C₁₅H₁₅NO₂F₂ calcd M279.1071, found M 279.10701.

Example 6

(1S, 4S)-6-Difluoromethylenyl-2-azabicyclo [2.2.1]heptan-3-one (14).Compound 13 (86.9 mg, 0.31 mmol) was dissolved in CH₃CN (1.75 mL). Asolution of ceric ammonium nitrate (512 mg, 0.93 mmol) in water (0.87mL) was slowly added. The resulting solution was stirred at roomtemperature for 4 h. The reaction mixture was then diluted with ethylacetate (20 mL), washed with brine (2×10 mL), and dried over anhydrousNa₂SO₄. After being concentrated under reduced pressure, the residue waspurified by flash column chromatography, eluting with hexanes/ethylacetate (1:1) to give the desired product as a colorless oil (33.6 mg,68%). ¹H NMR (400 MHz, CDCl₃) δ 5.48 (br s, 1H), 4.40 (s, 1H), 2.93 (s,1H), 2.54 (dd, J 15.2, 2.8 Hz, 1H), 2.32 (d, J 15.2 Hz, 1H), 2.15 (d, J9.6 Hz, 1H), 1.64 (d, J 10.0 Hz, 1H); ¹⁹F NMR (376 MHz, CDCl₃) δ 42.85and 40.00 (2d, J 60.2 Hz, 2F); HRMS (EI) C₇H₇NOF₂ calcd M 159.0496,found M 159.04673.

Example 7

(1S, 3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic acid (15). Tolactam 14 (20.0 mg, 0.13 mmol) was added 4 mL of 4 N HCl. The solutionwas stirred at 70° C. for 10 h. After being washed with ethyl acetate(3×4 mL), the water layer was evaporated under reduced pressure to givea yellow solid. Recrystallization with ethanol/ether gave a white solid,which was then loaded on a cation-exchange column (AG50W-X8) and elutedwith 0.2 N ammonium hydroxide to give the free amino acid 15 as a whitesolid (16 mg, 72%). ¹H NMR (400 MHz, D₂O) δ 4.44 (s, 1H), 2.92 (m, 1H),2.74 (m, 1H), 2.57 (dd, J 16.4, 3.6 Hz, 1H), 2.34 (m, 1H), 2.02 (d, J14.8 Hz, 1H); ¹³C NMR (126 MHz, D₂O) δ 186.08, 155.30 (t, J 288.7 Hz),92.19 (m), 53.16 (d, J 3.8 Hz), 48.01, 37.89, 32.45; ¹⁹F NMR (376 MHz,D₂O) δ −8.43 and −9.02 (2d, J 46.3 Hz, 2F); MS (ESI) C₇H₉NO₂F₂ calcd M+H178, found M+H 178.

Example 8

(E/Z)-(1S,4S)-6-(1′-Fluoro-1′-phenylsulfonyl)methylenyl-2-(4′-methoxybenzyl)-2-azabicyclo[2.2.2]heptan-3-one(16). To anhydrous THF (3 mL) was added fluoromethyl phenylsulfone (130mg, 0.75 mmol) and diethyl chlorophosphate (0.11 mL, 0.74 mmol). Aftercooling to −78° C. under nitrogen, lithium bis(trimethylsilyl)amide (1.0M in THF, 1.65 mL, 1.65 mmol) was slowly added. After stirring for 1 h,a solution of 12 (91.3 mg, 0.37 mmol) in anhydrous THF (3 mL) was slowlyadded via cannula. The solution was then warmed to room temperature andstirred overnight. After being quenched with saturated NH₄Cl solution(10 mL), THF was evaporated and the resulting solution was extractedwith ethyl acetate (3×10 mL). The organic layer was combined, washedwith brine, and dried over anhydrous Na₂SO₄. This solution was thenconcentrated under reduced pressure and purified with flash columnchromatography, eluting with hexanes/ethyl acetate (1:0 to 1:2), givingan inseparable cis/trans mixture (16) (4.4:1 as seen from NMR, 119 mg,80%) as a colorless oil. ¹H NMR for the major product (400 MHz, CDCl₃) δ7.94 (d, J 8.0 Hz, 2H), 7,72 (t, J 7.4 Hz, 1H), 7.61 (t, J 7.6 Hz, 2H),7.33 (d, J 8.4 Hz, 2H), 6.90 (d, J 8.8 Hz, 2H), 5.24 (s, 1H), 4.77(d, J14.8 Hz, 1H), 3.82 (s, 3H), 3.79 (d, J 14.8 Hz, 1H), 3.00 (s, 1H),2.49-2.66 (m, 2H), 2.10 (d, J 9.2 Hz, 1H), 1.63 (d, J 8.8 Hz, 1H).

Example 9

(E)-(1S,4S)-6-Fluoromethylenyl-2-(4′-methoxybenzyl)-2-azabicyclo[2.2.2]heptan-3-one(17) and(Z)-(1S,4S)-6-fluoromethylenyl-2-(4′-methoxybenzyl)-2-azabicyclo[2.2.2]heptan-3-one(18). Compound 16 (100 mg, 0.25 mmol) was dissolved in anhydrousmethanol (10 mL) under nitrogen and put in an ice-salt bath. Magnesiumturnings (0.30 g, 12.5 mmol) and mercury (II) chloride (60 mg, 0.22mmol) were added. The solution was stirred for 2 h, then warmed to roomtemperature and stirred overnight. The reaction mixture was poured into1 N HCl (10 mL). Methanol was evaporated under reduced pressure and theresulting water solution was extracted with ethyl acetate (3×10 mL). Theorganic layer was combined, washed with saturated NaHCO₃ solution (2×10mL), brine (2×10 mL), and dried over anhydrous Na₂SO₄. Afterconcentration under reduced pressure, the residue was purified by columnchromatography with hexanes/ethyl acetate (3:1), giving compound 17(33.8 mg, 52%) and 18 (12.9 mg, 20%), both as colorless oils.

For 17: ¹H NMR (500 MHz, CDCl₃) δ 7.15 (d, J 8.5 Hz, 2H), 6.87 (d, J 8.5Hz, 2H), 6.65 (d, J 82.9 Hz, 1H), 4.66 (d, J 15.0 Hz, 1H), 3.83 (s, 1H),3.81 (s, 3H), 3.72 (d, J 15.0 Hz, 1H), 2.98 (s, 1H), 2.55 (dd, J 16.0,2.5 Hz, 1H), 2.36 (dd, J 16.0, 1.5 Hz, 1H), 2.02 (d, J 8.0 Hz, 1H), 1.53(d, J 9.5 Hz, 1H).

For 18: ¹H NMR (500 MHz, CDCl₃) δ 7.21 (d, J 8.5 Hz, 2H), 6.87 (d, J 8.5Hz, 2H), 6.54 (d, J 84.9 Hz, 1H), 4.67 (d, J 15.0 Hz, 1H), 4.36 (s, 1H),3.81 (s, 3H), 3.67 (d, J 15.0 Hz, 1H), 2.96 (s, 1H), 2.43 (d, J 14.0 Hz,1H), 2.21 (d, J 15.0 Hz, 1H), 1.97 (d, J 9.5 Hz, 1H), 1.48 (d, J 9.5 Hz,1H).

Example 10

(E)-(1S,4S)-6-Fluoromethylenyl-2-azabicyclo[2.2.2]heptan-3-one (19). Inan Eppendorf tube, 17 (10.2 mg, 39 μmol) was dissolved in acetonitrile(0.22 mL). To this solution was added a solution of ceric ammoniumnitrate (64 mg, 117 μmol) in water (60 μL). After being stirred at roomtemperature for 3 h, the reaction mixture was diluted with ethyl acetate(10 mL), washed with brine (2×5 mL), and dried over anhydrous Na₂SO₄.After concentration under reduced pressure, the residue was purified bycolumn chromatography, eluting with hexanes/ethyl acetate (1:1) to give19 as a colorless oil (2.0 mg, 36%). ¹H NMR (400 MHz, CDCl₃) δ 6.83 (d,J 83.2 Hz, 1H), 5.48 (br s, 1H), 4.15 (s, 1H), 2.90 (s, 1H), 2.60 (d, J16.8 Hz, 1H), 2.39 (d, J 15.6 Hz, 1H), 2.15 (d, J 9.2 Hz, 1H), 1.61 (d,J 9.2 Hz, 1H); ¹⁹F NMR (376 MHz, CDCl₃) 6-2.75 (d, J 83.6 Hz, 1F).

Example 11

(E)-(1S, 4S)-6-Fluoromethylenyl-2-azabicyclo[2.2.2]heptan-3-one (21). ¹HNMR (400 MHz, CDCl₃) δ 6.47 (d, J 85.6 Hz, 1H), 5.40 (s, 1H), 4.61 (s,1H), 2.89 (s, 1H), 2.47 (d, J 14.8 Hz, 1H), 2.26 (d, J 16.0 Hz, 1H),2.13 (d, J 9.2 Hz, 1H); ¹⁹F NMR (376 MHz, CDCl₃) δ −0.27 (d, J 84.0 Hz,1F).

Example 12

(E)-(1S, 3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic acid,hydrochloride salt (20). To compound 19 (2.0 mg, 14 μmol) was added 4 NHCl (4 mL). The solution was heated to 70° C. and stirred for 10 h. Thenit was cooled, washed with ethyl acetate (2×4 mL), and evaporated underreduced pressure to give a white solid (2.0 mg, 72%). ¹H NMR (400 MHz,D₂O) δ 6.93 (d, J 81.2 Hz, 1H), 4.33 (m, 1H), 3.06 (t, J 8.0 Hz, 1H),2.91 (m, 1H), 2.71 (m, 1H), 2.48 (t, J 6.8 Hz, 1H), 2.03 (t, 6.8 Hz,1H); ¹⁹F NMR (376 MHz, D₂O) δ −48.59 (d, J 78.7 Hz, 1F).

Example 13

(Z)-(1S, 3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic acid,hydrochloride salt (22). ¹H NMR (400 MHz, D₂O) δ 6.82 (d, J 82.4 Hz,1H), 4.50 (s, 1H), 3.00 (p, J 8.0 Hz, 1H), 2.70 (m, 1H), 2.48-2.62 (m,2H), 1.99 (m, 1H); ¹⁹F NMR (376 MHz, D₂O) δ −50.47 (d, J 82.5 Hz, 1F).

Example 14a

Time-dependent Inactivation of GABA-AT by 15 and (S)-vigabatrin.Incubation solutions (100 μL) contain commercially available GABA-AT (20μL, 1.84 mg/mL, specific activity 2.5 unit/mg), potassium pyrophosphatebuffer (60 μL, 50 mM, pH 6.5), α-ketoglutarate (10 μL, 16 mM in 50 mMpotassium pyrophosphate buffer, pH 6.5), 2-mercaptoethanol (2 mM), and15 or (S)-vigabatrin (10 μL, with varied concentrations of compounds in50 mM potassium pyrophosphate buffer, pH 6.5). The concentrations for 15and vigabatrin are as follows: 66.8, 74.1, 95.5, 111.2 μM for 15 and334, 417.5, 477.0, 556.4, 668 mM for (S)-vigabatrin. At timed intervals(about one minute for 15 and three minutes for (S)-vigabatrin), aliquots(20 μL) from the incubation solution were added to the assay solution(575 μL, 50 mM potassium pyrophosphate buffer containing 5.3 mM ofα-ketoglutarate, 11 mM of GABA, 1.1 mM of NADP⁺ and 4.8 mM of2-mercaptoethanol) with excess SSDH. Rates were measuredspectrophotometrically at 340 nm, and the logarithm of the remainingactivity (percentage) was plotted against time for each concentration todetermine the half-life. Then a secondary plot of half-life versus thereciprocal of inactivator concentration was obtained to determine K_(I)and k_(inact).

Example 14b

At pH 8.5 and 25° C. the specificity constant (k_(inact)/K_(I)) for thedifluoromethylene inactivator is 186 times that for vigabatrin (seeTable 1 below).

TABLE 1 Difluoromethylene Inactivator Vigabatrin K₁ = 9.7 μM K₁ = 850 μMk_(inact) = 0.50 min⁻¹ k_(inact) = 0.24 min⁻¹ k_(inact)/K₁ = 0.052μMmin⁻¹ k_(inact)/K₁ = 0.00028 μMmin⁻¹

Example 14c

Using experimental protocols such as those provided in example 14a,compounds 20 and 22 are used effectively to inhibit GABA-AT. Comparableresults are obtained with the trans isomer.

Example 15

Studies were undertaken to characterize the addition product of GABA-ATand an inhibitor compound of this invention. To that effect, utilizingprocedures analogous to that described in example 14a, two incubationsolutions were prepared, one with 543 equiv. of a difluoro-substitutedcompound and another with 1 equiv. of the same compound. Each wasmonitored for fluoride ion using an Orion 720A pH meter and an Orion96-09 fluoride/combination fluoride electrode. (A standard curve wasmade each time before measuring fluoride concentration.)

In the presence of 543 equiv. of the difluoro compound, the fluoride ionconcentration was 1.07 equiv. after inactivation, 4.4 equiv. after 24hours, and 4.9 equiv. after 48 hours. With 1 equiv. of the subjectdifluoro inactivator compound, fluoride ion concentration was 0.75equiv. at 15% enzyme activity (about 1 hour, 33 minutes) and 1.55 equiv.at 24 hours.

The data of this example show that one equiv. of fluoride ion isreleased from the subject inactivator compound after complete loss ofenzymatic activity, but that one or more additional fluoride ions arelost non-enzymatically thereafter, slowly over time, possibly from ametabolite of the addition product complex.

1. A composition comprising a γ-aminobutyric acid aminotransferaseinhibitor compound selected from compounds of a formula

wherein R₁ and R₂ are selected from H and F, and at least one of R₁ andR₂ is F; and salts thereof.
 2. The composition of claim 1 wherein R₁ andR₂ are F.
 3. The composition of claim 2 wherein said NH₂ and COOHsubstituents have a stereochemical relationship selected from cis andtrans.
 4. The composition of claim 3 wherein said substituents are cis.5. The composition of claim 2 selected from an ammonium salt and acarboxylate salt of said compound.
 6. The composition of claim 5 whereinsaid compound is an ammonium salt, and the counter ion is the conjugatebase of a protic acid.
 7. The composition of claim 5 wherein saidcompound is a carboxylate, and the counter ion is selected from theconjugate acid of an amine, alkaline and alkaline-earth base.
 8. Thecomposition of claim 5 wherein said compound is selected from anammonium hydrochloride salt and a sodium carboxylate.
 9. The compositionof claim 1 wherein one of said R₁ and R₂ is F.
 10. The composition ofclaim 9 wherein R₁ is F, and said F and COOH substituents have a Zconfiguration.
 11. The composition of claim 10 wherein said NH₂ and COOHsubstituents have a stereochemical relationship selected from cis andtrans.
 12. The composition of claim 9 wherein R₁ is F, and said F andCOOH substituents have an E configuration.
 13. The composition of claim12 wherein said NH₂ and said COOH substituents have a stereochemicalrelationship selected from cis and trans.
 14. The composition of claim 9selected from an ammonium salt and a carboxylate salt of said compound.15. The composition of claim 1 wherein said compound is in a fluidmedium.
 16. The composition of claim 15 contacting a γ-aminobutyric acidaminotransferase.
 17. The composition of claim 16 wherein said compoundis in an amount at least partially sufficient for inhibition of saidtransferase.